Project Summary/Abstract Excessive production of glucose by the liver plays a central role in the development of diabetes, where the ability of insulin to regulate hepatic glucose production (HGP) is impaired. FoxO transcription factors are major targets of insulin and play an important role in mediating effects of insulin on multiple aspects of glucose and lipid metabolism in the liver, including gluconeogenesis, glycolysis and lipogenesis. However, the mechanisms by which FoxO proteins exert these diverse effects in an integrated fashion remain poorly understood. During our previous funding period, we found that a) FoxO proteins also exert important effects on intrahepatic lipolysis and fatty acid oxidation via the regulation of adipose triacylglycerol lipase (ATGL), which mediates the first step in lipolysis, and its inhibitor, the G0/S1 switch 2 gene (G0S2), and b) ATGL-dependent lipolysis plays a critical role in mediating diverse effects of FoxO proteins in the liver, including effects on gluconeogenic, glycolytic and lipogenic gene expression and metabolism. These studies also indicate that ATGL-dependent lipolysis is required to mediate effects of FoxO1 on glycolytic and gluconeogenic gene expression, and additional studies are planned in mouse models and isolated hepatocytes to better understand specific mechanisms mediating this novel link between lipid metabolism and glucoregulation by the insulin/FoxO pathway. In addition, using liver-specific insulin receptor (IR) knockout (LIRKO) and IR/FoxO1 double knockout (LIRFKO) mice, we also found that disrupting FoxO1 in the liver was sufficient to restore the ability of insulin to maintain glucose homeostasis and suppress HGP (based on euglycemic hyperinsulinemic clamp studies) in mice lacking the hepatic insulin receptor. These results indicate that (a) inhibition of FoxO1 is critical for both direct (hepatic) and indirect effects of insulin on HGP and glucose utilization, and (b) extrahepatic effects of insulin are sufficient to maintain normal whole-body and hepatic glucose metabolism when liver FoxO1 activity is disrupted. Based on these findings, additional studies utilizing transgenic and knockout mouse models, cell culture and insulin clamp techniques are planned to determine whether ATGL- dependent lipolysis also plays a critical role in mediating effects of FoxO1 on hepatic glucose metabolism in the setting of hepatic insulin resistance, and whether targeting ATGL and its downstream effectors may provide an effective strategy for treatment of diabetes mellitus in patients with hepatic insulin resistance.